Radioimmunoassay (RIA) methods generally fall into two categories: liquid and solid phase. Liquid phase methods, which involve immune complexes in the form of precipitates suspended in a liquid phase, have been widely used for detection and quantitation of protein containing antigens, including hormones and viruses. However, solid phase methods are enjoying more widespread use, particularly for detecting and quantitating viral antigens and antibodies. The solid phase may involve surface areas in test tubes (U.S. Pat. No. 3,867,517), in depression plates, on beads, or any other convenience device (U.S. Pat. No. 3,826,619) whose surfaces are of a composition which will adsorb the antigen or antibody reactants and which are of a size and shape which will fit into a gamma counter (see also Journal of Immunological Methods, Vol. 5 (1974) pp. 337-344). Advantages in using solid phase RIA systems include the capability for efficient rinsing and transferring of the solid phase, with the antigen-antibody complexes on its surface, without the requirement for centrifugation-resuspension of antigen-antibody precipitates required in most liquid phase methods.
Spherical beads are particularly useful as solid phases (see Journal of Clinical Microbiology, August 1975, pp. 130-133) because beads can be obtained with extremely uniform diameters (hence surface areas); they each contact the bottom of a container at only one small point, they can be totally immersed in reagent solutions and, for these reasons, give highly reproducible results in RIA procedures. Such precision is difficult to obtain with inner surfaces of test tubes or depression plate wells, probably because: reactants adsorb to the surfaces of these vessels to various heights during slight "sloshing" attendant with movements of the vessels; splashing of reagents sequentially added during the test and streaming of reagents down the vessels' sides occur during their delivery. Therefore, unfortunate variations in the surface areas exposed to reagents are inevitable. Immersed beads, on the other hand, allow high uniformity of surface areas exposed to reagents and, when totally immersed, exposure of this uniform surface is highly reproducible and results in high RIA precision. As examples of the superiority of the bead solid phase system for RIA over tube solid phase systems, the test for Australia antigen has been heretofore converted from the tube to the bead test, and bead tests are the only ones currently licensed for use in the United States for detection of hepatitis B antigen in blood donors' sera.
Processing and transferring beads by the presently used methods, however, leaves much to be desired. The use of forceps (page 339, Journal of Immunological Methods, supra) to transfer each bead separately is tedious, time consuming and subject to inadvertent small variations inevitable when handling is manual. These deficiencies significantly limit the usefulness of bead assay methods because: (1) the number of assays which can be done at one time under reasonably identical conditions of timing is restricted; (2) small variations in manual handling, especially between different technicians, limit the precision with which assays can be accomplished and may be one of the precision limiting factors in the test; and (3) the cost associated with tedious manual manipulation of many individual beads is substantial.
These problems have been partially solved in a design of a test for hepatitis B antigen by allowing beads to remain in the same depression plate wells throughout the RIA procedure, merely aspirating reagents and rinsing beads in many wells simultaneously with a mechanically sophisticated, manifold type rinsing-dispensing device, then dumping the beads by the force of gravity from the depression plate wells into tubes for insertion into a gamma counter. There are some objections to this procedure, however, when considering optimal test conditions for the hepatitis test and other applications in particular. Performing a RIA bead test completely in one well involves reactions with the well's walls simultaneously with the bead reaction. In effect, then, the well wall surface competes with the bead surface for reagents introduced later in the test. Non-specific adsorption of reactants is usually followed by a small amount of release of reactants into succeeding reagents added, which is unfortunately magnified quantitatively if the same well is used throughout the test. Transferring the beads to new wells during the test with facility would obviate this problem. Aspiration of fluids from small volume cul de sacs containing beads with large surface areas, followed by addition of small volumes of rinsing fluid nevertheless constitutes a relatively inefficient rinsing procedure because of the difficulty in aspirating all of the fluid in the well (a small cul de sac containing an object with a maximum surface area, a sphere, hence attracting maximum fluid volume due to capillary or surface tension effects). Also, this approach does not eliminate the necessity for transferring beads one-at-a-time by forceps into the wells initially. If there are dozens of wells and a short incubation period, treatment of the first and last beads will necessarily be significantly different. Similarly, exact volumes of new reagents must be delivered to each separate well during the RIA process. Thus, beads exposed first during this procedure will not be treated identically to beads exposed at the last of the dispensing period. Therefore, when large numbers of beads are employed in an assay run (as when testing many different specimens) and short reaction periods are desirable (30 minutes, for example), uniformity in test conditions is seriously compromised and the precision of the assay suffers substantially. Inability to expose all beads to the same reagents simultaneously thus limits the usefulness of any RIA procedure, but particularly those procedures involving many specimens, short incubation times and numerous reagents.